【作者】 闫华晓；

【导师】 崔德才；

【作者基本信息】 山东农业大学 ， 生物化学与分子生物学， 2003， 硕士

【摘要】 草莓 (Fragaria ananassa Duch.) 是一种经济价值较高的小浆果，为一种多年生草本植物。因为成熟期比较集中，储存期较短，所以限制了其运输和大规模生产。本研究课题以草莓为实验材料，以其叶片为外植体，系统地研究了激素浓度、叶片生理状态、光照条件等诸多因素对不定芽再生的影响，建立了草莓叶片培养诱导不定芽高频再生的体系。在此基础上，用携带反义磷脂酶Dγ基因（PLDγ）的根癌农杆菌介导转化草莓叶片，通过对转化方法和转化条件的探索，建立了草莓的遗传转化体系。草莓遗传转化体系的建立对利用基因工程对草莓进行品种改良起着重要的作用。研究结果表明不同基因型、叶龄、暗培养、基本培养基、不同激素组合、Ag+等诸多因素对叶片再生有显著的影响，建立了不依赖于TDZ的弗吉尼亚草莓叶片外植体的不定芽再生体系。研究结果表明Ag+ 并不能促进草莓弗吉尼亚植株的再生，反而起到了抑制作用。以顶部充分伸展的18—21天叶龄的无菌苗叶片为外植体，再生效果最好，在叶片远轴面上划几刀，造成伤口，以该面接触培养基，接种在以MS为基本培养基、附加6-BA 1.6-1.8mg/L, 6-BA 0.1mg/L NAA, KT 0.4-0.5mg/L,和2,4-D 0.1mg/L的分化培养基上诱导芽分化，14天后分化出不定芽，不定芽出现的高峰期在接种后的20-30天。通常不定芽散布在叶片的各个部位，以切口部位较多，芽分化率高达60%以上。该再生体系可作为基因转化的受体系统。在建立了草莓叶片培养诱导芽高频再生体系的基础上，建立并优化了农杆菌介导的草莓遗传转化体系。具体过程是：将培养至对数生长期的农杆菌 (OD600=0.5) 用等体积的5%的蔗糖溶液（含一定浓度的表面活性剂）稀释成侵染液，侵染叶片10分钟，将侵染之后的叶片置于含上述蔗糖溶液的<WP=6>培养皿中，共培养3天。用无菌蒸馏水冲洗叶片3次，滤纸吸干后接种于含300mg/L Cef的分化培养基上，恢复培养7天。最后转到选择培养基上(MS+1.6-1.8mg/L6-BA+0.1mg/LNAA+0.4-0.5mg/LKT+0.1mg/L2,4-D+300mg/L Cef+20mg/L Kan)培养，筛选抗性苗。对抗性植株进行PCR检测和PCR-Southern杂交检测，结果表明反义PLDγ基因已整合到植物基因组中。更多还原

【Abstract】 Strawberry( Fragaria ananassa Duch.) is a kind of small，perennial berry with high economic worth .Because the mature period is concentrate, its conveyance with the large-scale production are limited. The leaves of strawberry were used as the material and several factors such as hormone concentration, physiological conditions of explant, illumination of culture , and so on were investigated to optimize the regeneration system in vitro. A high frequency regeneration system in culture has been etablished. This culture system was adapted to Agrobacerium-mediated transformation. The young leaf of strawberry were inoculated with Agrobacerium tumefaciens strains harboring binary vectors, which contained the neomycin phosphotransferase Ⅱ gene and the anti-phospholipase Dγ(PLDγ)gene. The establishing of strawberry heredity converts system plays an important role in improving strawberry species by making use of the genetic engineering.The research result expresses the different genotype, leaf’s age , browness, darkness to culture, MS medium , different hormone , Ag+ and so on have influenced leave regeneration. The results showed that 6-BA and 2,4-D in plant initiation medium played an important role in improving plant regeneration. The most effective concentration for 6-BA and 2,4-D was 1.6-1.8mg/L, 0.1mg/L, which increased the regeneration frequency of Tudla(Fragaria×ananassa) to 60%. The regeneration frequency of Tudla was<WP=8>decreased by applying AgNO3 to the medium. The best explants are those apical leaves which from the 18-21 days cultured plantlets. Leaves were placed on the medium which is mentioned above for shoot induction. After 14 days cultured , the explants began to directly regenerate adventitious buds. The maximum of the adventitious buds were observed within 20 to 30 days. The shoot differentiation frequency was more than 60%. On the basis of regeneration system, transformation system was established by screening transformation conditions with antibiotics selection. Diluted the Agrobacterium until the OD(600nm) ranging from 0.3 to 0.5 with the same volume of 0.5% sucrose solution (contain concentration detergent), inoculated the strawberry leaves for 10 minutes, then co-cultivated the explants on sucrose liquid medium for 3 days and placed on the regeneration medium with 300mg/L Cef for 7 days. The resistant shoots were obtained on regeneration medium with 300mg/L Cef and 20mg/L Kanamycin.The presence and integration of the antisense PLDγ gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis.更多还原